Recombinant DNA techniques in which transfection vectors are inserted into cells are used to express genes for experimental study, gene replacement and the production of commercial products, such as, proteins, enzymes, and hormones. It is often desirable to have a selectable marker for identification of cells into which the transfection vector has been inserted. Presently, most DNA transfection vectors have as part of their gene code dominant selectable markers which provide the transfected cells, which are the cells into which the transfection vectors have been inserted, resistance to drugs or nutrient-depleted growth medium so that the transfected cells can be selected using the drug or the absence of the nutrient in the growth medium to kill the cells that do not have the transfection vector. Examples of these DNA transfection vectors are reviewed in Kriegler, M., Gene Transfer and Expression a Laboratory Manual, W. H. Freeman and Company, New York (1990).
Most methods of selecting transfected cells which require the use of drugs or nutrient-depleted growth mediums to select the cells are laborious and time consuming. For example, Wigler et al., Cell, Vol. 11, pg. 223 (1977) describe a method in which a transfection vector having the herpes virus thymidine kinase (TK) gene is inserted into cultured mouse cells which do not originally have the TK gene. The TK gene confers resistance to the drug aminopterin. In addition to requiring a special growth medium (HAT medium), the drug aminopterin is required to select the transfected cells. This selection method takes several days.
Another example of a commonly utilized selectable marker in a transfection vector is the aph gene which encodes resistance to the drug G418.TM. supplied by Life Technologies, Inc. This gene and the method of its use to select transfected cells is described in Southern, P. et al., J. Mol Appl. Gen. Vol. 1, pg. 327 (1982). To select the transfected cells with the aph gene a titration requiring several days must be performed on the cell sample prior to the introduction of the transfection vector, and after the introduction of the transfection vector to the cell sample the concentration level of G418.TM. must be maintained at the maximum tolerable drug level for several days to kill the nontransfected cells. This long process can be deleterious to the desired transfected cells.
It is commonplace to use toxic drugs as the means of selecting transfected cells; however, the toxic drugs can be toxic to the transfected cells or they can retard the growth of the transfected cells.
Padmanabhan, et al., Methods in Enzymology, Vol. 218, pp. 637-651 (1993) describe genes for naturally-occurring cell surface markers. Two examples of the naturally occurring cell surface markers are the vesicular stomatitis virus glycoprotein (VSV-G) and the multiple drug resistance marker (mdr). Transfected cells which express the gene for naturally-occurring cell surface markers are identified using antibodies which are specific to the cell surface marker. Genes expressing naturally-occurring cell surface markers are deficient, because they have inherent biological properties which may interfere with normal cell function. Also, most naturally-occurring cell markers are large proteins whose genes take up more space in a tranfection vector, are harder to work with, are prone to having unwanted restriction enzyme sites and are more of a protein synthesis burden on the transfected cell.
There is therefore presently a need in the art for genes which express selectable markers which can be used in transfection vectors for the identification and selection of transfected cells, which are not naturally occurring markers, do not require laborious and time consuming techniques, and do not use toxic drugs to identify the transfected cells.